A 28-year-old man from Delhi with a family history of cystic fibrosis (CF) presents for carrier screening before marriage. His sister was diagnosed with CF due to a known CFTR gene deletion (ΔF508). The genetic counsellor orders molecular testing to determine if he carries the same mutation. Which combination of techniques would most efficiently detect the ΔF508 deletion and simultaneously identify any additional CFTR mutations in this patient?

Explanation

## Molecular Screening for Cystic Fibrosis Carrier Status ### Clinical Context Cystic fibrosis is an autosomal recessive disorder caused by mutations in the CFTR gene. ΔF508 (deletion of phenylalanine at codon 508) is the most common mutation worldwide (~70% of CF alleles). Carrier screening requires detection of both known mutations (like ΔF508) and unknown variants. ### Why Multiplex PCR + Sequencing? **Key Point:** Multiplex PCR amplifies multiple CFTR regions in a single reaction, followed by direct DNA sequencing to identify both the known ΔF508 deletion and any additional or novel mutations. **High-Yield:** This two-step approach combines: 1. **Efficiency**: Multiplex PCR amplifies 10–20 common CFTR mutation hotspots simultaneously. 2. **Sensitivity**: Direct sequencing of PCR products detects point mutations, small indels, and known deletions. 3. **Completeness**: Identifies both the expected ΔF508 and any unexpected mutations, improving diagnostic yield. ### Detection of ΔF508 Deletion **Clinical Pearl:** ΔF508 is a 3-bp in-frame deletion (CTT) at codon 508. Multiplex PCR primers flanking this region will produce: - **Wild-type allele**: 1,272 bp (or expected size) - **ΔF508 allele**: 1,269 bp (3 bp shorter) Direct sequencing of both bands confirms the deletion at the nucleotide level. ### Comparison of Techniques for CFTR Mutation Detection | Technique | Detects | Limitations for CF Screening | |-----------|---------|------------------------------| | **Multiplex PCR + Sequencing** | Point mutations, small indels, known deletions | **Best for carrier screening** | | **Southern blotting** | Large deletions, rearrangements | Slow, labor-intensive, misses point mutations | | **Northern blotting** | mRNA abnormalities, splicing defects | Requires RNA; cannot detect DNA mutations; tissue-dependent | | **qRT-PCR** | Gene expression, copy number | Detects dosage, not sequence mutations | | **AFLP (Amplified Fragment Length Polymorphism)** | Polymorphisms, deletions | Non-specific; requires multiple digestions | ### Diagnostic Algorithm for CF Carrier Screening ```mermaid flowchart TD A[CF carrier screening requested]:::outcome --> B[Multiplex PCR of CFTR hotspots]:::action B --> C[Amplify 10-20 common mutation regions]:::action C --> D[Direct DNA sequencing of PCR products]:::action D --> E{ΔF508 detected?}:::decision E -->|Yes| F[Heterozygous carrier confirmed]:::outcome E -->|No| G{Other mutations found?}:::decision G -->|Yes| H[Identify mutation; assess pathogenicity]:::action G -->|No| I[Negative for common mutations; consider full gene sequencing]:::action H --> J[Genetic counselling: carrier status, reproductive risk]:::outcome I --> J ``` **Mnemonic:** **M-PCR-SEQ** = **Multiplex PCR followed by SEQuencing** — the efficient, high-yield approach for detecting both known and novel CFTR mutations in carrier screening. ### Why Not the Other Options? **Southern Blotting Limitation:** - Detects large deletions and rearrangements, but ΔF508 is only 3 bp and requires very high resolution. - Slow (days to weeks); requires radioactive probes or chemiluminescence. - Misses point mutations entirely. **Northern Blotting Limitation:** - Detects mRNA abnormalities and splicing defects, not genomic DNA mutations. - Requires fresh tissue (lymphocytes, fibroblasts); not practical for carrier screening. - Cannot detect ΔF508 directly. **qRT-PCR Alone Limitation:** - Measures gene expression and copy number, not sequence mutations. - A carrier with one normal and one ΔF508 allele will have ~50% CFTR expression, but qRT-PCR alone cannot identify the mutation. - Requires a second confirmatory test.